- Title
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The knockdown efficiency of telomere associated genes with specific methodology in a zebrafish cell line
- Authors
- Hu, X., Gao, S., Wang, P., Zhou, Y., Chen, K., Chen, Q., Wang, B., Hu, W., Cheng, P., Eid, R., Giraud-Panis, M.J., Wang, L., Gilson, E., Ye, J., Lu, Y.
- Source
- Full text @ Biochimie
Fig. 1. Example for a schematic layout of optimization strategies. 1–2 × 105 ZF4 cells were seeded in 24-well plates. Transfection experiments were proceeded at a final confluence of 50%–70%. Numbers in each well refers to trial-1–3 of each transfection reagent. All transfection methods of an optimization experiment were proceeded at the same time. Optimization was repeated three times for individual experiments. |
Fig. 2. Vivo-MOs mediated gene knockdown targeting terfa in ZF4. A. zfTRF2 expression of each trial by Western blot. B. Expression of zfTRF2 in ZF4 cells 48, 72 and 96 h after being treated with 7.5 μM vivo-MOs. C. Microscopy of ZF4 cells 72 h after being treated with 7.5 μM vivo-MOs. Ns: none sense, ∗: p < 0.05, scale bar: 50 μm. |
Fig. 3. SiRNAs mediated gene knockdown targeting terfa in ZF4. A.B. Knockdown efficiency of terfa of each scheme by RT-qPCR. A, 48 h after transfection; B, 72 h after transfection. C. Western blot of zfTRF2. Untreated and siRNAs transfected cells were prepared for WB after being transfected with X–HP for 72 h. D. SiRNAs mediated gene knockdown in ZF4 and ZFL. Knockdown efficiency of terfa was measured by RT-qPCR 48 h after transfection with 15 μL X-tremeGENE HP and 400 nM siRNAs in 6-well plate. E. SA-β-Gal assay of ZF4 transfected with siRNAs following trial-3 of X–HP at 72 h. Scale bar: 20 μm. F. Microscopy of untreated and transfected ZF4 cells. Results showed little cytotoxicity of Lipo3000 and X–HP and medium cytotoxicity of jetPrime, Effect a min and X-siRNA. scale bar: 50 μm. G. Off-target effect detection by RT-qPCR of shelterin genes terfa, terf1, pot1, rap1, tpp1, tin2 in ZF4 cells transfected with siterfa by X–HP 48 h later. Lipo3000: LipofectaminTM 3000, X-siRNA: X-tremeGENETM siRNA, Effectene: Effectene® Transfection Reagent, X-HP: X-tremeGENETM HP.ns: none sense, ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001. |
Fig. 4. SiRNAs targeting terf1, pot1, rap1, tpp1 and tin2 were synthesized and transfected into ZF4 cells by X-tremeGENETM HP following trail-3 in Table 1. For each gene, two siRNAs of different target sequences were transfected alone or together to reach higher knockdown efficiency. Relative mRNA level of each gene was measured by RT-qPCR 48 h after transfection, respectively. Ns: none sense, ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001. |